The rapid development of biotechnological methods for the manufacture of biomolecules, such as proteins and peptides, also put new requirements on the equipment used in such processes. For example, in the pharmaceutical industry, the end product must meet certain demands on purity and safety to be approved as a drug by the authorities. A process for preparation of biomolecules usually involves a number of purification steps, such as filtration, precipitation, chromatography etc. Liquid chromatography is a well known and much used technique, which may briefly be described as the fractionation of components of a mixture based on differences in the physical or chemical characteristics of the components. More specifically, in chromatography, two mutually immiscible phases are brought into contact wherein one phase, commonly known as the matrix or a resin, is stationary and the other one is mobile. The sample mixture, introduced into the mobile phase, undergoes a series of interactions between the stationary and mobile phases as it is being carried through the system by the mobile phase. Interactions which exploit differences in the physical or chemical properties of the components in the sample govern the rate of migration of the individual components moving through a column. Due to high versatility, chromatography is commonly used in biotechnological processing and any other method where it is desired to separate out one or more useful components from a fluid mixture that contains other components, which may not be useful or are less valuable. Thus, chromatography is useful e.g. in methods that involve removal and/or isolation of viruses, nucleic acids, pyrogens, fine chemicals, food additives, diagnostics and drugs.
A conventional process for the preparation of biomolecules, such as proteins and peptides, commonly includes an initial cleaning in place (cip) sanitization of vessels, filters, chromatography columns etc, commonly by a solution of sodium hydroxide, and subsequently operating while maintaining as low a bioburden as possible. To obtain a sterile product, very clean conditions are maintained until the last step, which involves passing the product through a sterile filter into a sterile container.
However, a well known problem of such a conventional process is that products having a distinct large three-dimensional structure, such as large DNA molecules, virus, and protein complexes, cannot be sterile filtered without impairing recovery. For example, commonly sterile filters have pore sizes of 0.22 μm, while e.g. vaccinia viruses, rod-like viruses, have diameters up to 260 nm and can be up to 700 μm long. Alternative ways of sterilizing the products are known, such as addition of chemicals or autoclaving. However, due to the high purity requirements, addition of chemicals is usually avoided in the biotechnical preparation of drug molecules. Further, autoclaving will involve temperatures and pressures which would readily kill living organisms, or at least make substantial changes to their conformation thus eradicating their biological activity, and is consequently not suitable for products such as virus and plasmids.
An alternative to the above-discussed sterilization of the end product would be to run the whole process starting from sterile raw materials, and using previously sterilized equipment. “Equipment” in this context would include fermenter, commonly containing the fermentation broth, tubings, other vessels, filters, centrifuges, chromatography columns and the like. One of the most frequently method used for sterilization of process equipment in general is autoclaving, and of the above, fermenters, optionally filled with broth, are commonly autoclaved without any serious problems, provided their size allows easy movement thereof. In general, tubings, vessels and filters are also easily autoclavable. A specific case of filters known as hollow fibres, wherein a thin layer of resin has been immobilised to the inside of a hollow fibre, have been sterilized with steam and/or hot water, but have also successfully been autoclaved. This is mainly because the layer of resin in a hollow fibre is thin enough not to impose any problems with regard to uniformity and packing properties.
However, the process of autoclaving is more complex with regard to the chromatography equipment. Firstly, in large scale processing, chromatography columns will often be of a size which is not readily moved into an autoclave. Secondly, the pressure within a closed vessel, such as a packed chromatography column, in an autoclave may impose serious problems, for example as regards uniformity and other properties of the packing. For that reason, the sterilization of chromatography columns and chromatography matrices are commonly carried out separately, which means that a subsequent step for packing the column will be required. In addition to being time consuming and hence costly, such a packing may be difficult to ensure in practise without contamination. Thirdly, due to the low heat transport in the resin during autoclaving, sterilization would require a very long treatment time, and will in some cases still not be properly achieved throughout the matrix.
An example of separate sterilization of different components is disclosed in U.S. Pat. No. 5,817,528 (Böhm et al), which relates to a method for producing a sterile and pyrogen-free column that contains coupled protein intended for use in removing a predetermined substance from the blood of a human subject. According to U.S. Pat. No. 5,817,528, sterilization of the finished protein-containing product is achieved by providing sterile and pyrogen-free raw materials at each production step. More specifically, the method provides a pathogen-free, purified protein solution; and a sterile and pyrogen-free column matrix material, such as an agarose. The sterile and pyrogen-free, activated matrix material and the pathogen-free, purified protein solution are then combined under aseptic conditions to effect the binding of the protein to the matrix material, and the protein-coupled matrix material is filled under aseptic conditions into a sterile and pyrogen-free housing to produce a sterile and pyrogen-free column. However, such a process will require a number of process steps, which is disadvantageous in an industrial process since each step will increase the total costs.
Sterilization of a packed chromatography column is disclosed in U.S. Pat. No. 5,423,982 (Jungbauer et al), which relates to a liquid chromatography column well-suited for in situ sterilization effected by washing with a sterilization solution. A specific arrangement in the chromatography column, including a multilayered sintered metal filter and a corrugated expanding ring at the outlet, is stated to reduce or eliminate the “dead spaces” where microbes can become secluded from sterilizing solutions. The sterilizing solution used in U.S. Pat. No. 5,423,982 on a packed bed contain 1500 ppm peracetic acid as sterilizing agent.
Another example of chemical sterilization is disclosed in U.S. Pat. No. 5,676,837 (Jungbauer et al), which relates to a method for sterilization liquid chromatography resins that are highly resistant to oxidation by strongly oxidizing agents. According to U.S. Pat. No. 5,676,837, one example of a commonly used sterilizing agent is ethanol/water at a neutral or acidic pH, a common concentration being about 20% ethanol. However, as is well known, 20% ethanol has no sporocidal effect, and is therefore not completely sterilizing, and in addition large molecular aggregates may be destabilized by such a treatment. U.S. Pat. No. 5,676,837 states that a commonly used sterilization technique for instruments and the like, namely destroying microbes by wet heat, has not been employed to sterilize chromatographic resins due to their common temperature sensitivity. To avoid drawbacks such as the above, U.S. Pat. No. 5,676,837 suggests a method of sterilizing a chromatography resin by washing it with an aqueous solution of a percarboxylic acid which solution also contains an acetate buffer in a concentration of about 0.1-2M. The sterilization can be performed in a separate vessel or, alternatively, by pouring the solution through the packed chromatography column.
In summary, it is concluded that there is still a need in this field of robust methods for sterilization including spore destruction of chromatography columns, in particular in the context of processes for the manufacture of large targets, such as viral vectors and plasmid DNA. More specifically, there is a need for a sterile packed chromatography column which is adaptable to a sterile process.